The spliceosome is a large, dynamic assembly of ribonucleoproteins that catalyzes intron removal from pre- messenger RNAs. Spliceosomal RNAs and proteins are critical for eukaryotic gene expression, yet little is understood about their structure and function. The goal of the proposed research is to elucidate the structures of highly conserved RNA complexes and ribonucleoproteins from the spliceosome, and understand how the structures change throughout the splicing cycle. Aim 1 is to investigate the structure and function of the U6 RNA-Prp24 ribonucleoprotein complex. Prp24 protein tightly binds to U6 RNA in the free U6 snRNP and is required for U6 RNA structural remodeling during spliceosome assembly and activation. We recently solved the crystal structure of the N-terminal two-thirds of Prp24, and defined a putative U6 RNA binding surface by NMR. The structure of U6 RNA will be determined alone and in complex with Prp24, using both NMR and X- ray studies. These data will reveal how Prp24 remodels the U6 RNA structure to facilitate pairing with U4 RNA during spliceosome assembly, and with U2 RNA during spliceosome activation. In vitro biochemical and in vivo genetic studies will be used to further probe the mechanism by which Prp24 acts as a U6 RNA chaperone. Aim 2 of the proposal is to determine the structure of the U2-U6 RNA complex. The U2-U6 complex resides within the catalytic core of the spliceosome and directly hydrogen bonds to the pre-mRNA substrate in the active site. Furthermore, the human U2-U6 complex has residual catalytic activity in the absence of proteins. For this aim, we have identified a well-folded fragment of U2-U6 RNA that maintains tertiary structure and is amenable to NMR structure determination. As a sub-aim, we will collaboratively develop new methods for refining large, multi-domain RNA NMR structures against small angle X-ray scattering (SAXS) data and pseudo-CSA restraints. We expect these methods will be of general utility for structure determination of large macromolecules in solution. Prp24 binds to the U2-U6 RNA construct and we will attempt to determine the structure of this ribonucleoprotein complex, which may mimic a true intermediate in spliceosome activation and/or disassembly. These studies will significantly advance our understanding of the allosteric cascade that drives spliceosome assembly, activation, catalysis and disassembly.Narrative The spliceosome is an intricate cellular machine composed of 5 RNAs and more than 75 proteins. The spliceosome is so vital to the cell that even a subtle defect in just one of its components results in the disease retinitis pigmentosa, which causes blindness. Our work will result in a better understanding of the mechanism by which this remarkable molecular machine processes messenger RNA so that proteins can be made correctly by cells.